1. Field of the Invention
The present invention relates to conjugate vaccines. More particularly, the present invention relates to solid phase conjugate vaccines.
2. Description of Related Arts
In the process of vaccination, medical science uses the body""s innate ability to protect itself against invading agents by immunizing the body with antigens that will not cause the disease but will stimulate the formation of antibodies that will protect against the disease. For example, dead organisms are injected to protect against bacterial diseases such as typhoid fever and whooping cough, toxins are injected to protect against tetanus and botulism, and attenuated organisms are injected to protect against viral diseases such as poliomyelitis and measles.
It is not always possible, however, to stimulate antibody formation merely by injecting the foreign agent. The vaccine preparation must be immunogenic, that is, it must be able to induce an immune response. Certain agents such as tetanus toxoid are innately immunogenic, and may be administered in vaccines without modification. Other important agents are not immunogenic, however, and must be converted into immunogenic molecules before they can induce an immune response. The immune response is a complex series of reactions that can generally be described as follows: (1) the antigen enters the body and encounters antigen-presenting cells which process the antigen and retain fragments of the antigen on their surfaces; (2) the antigen fragments retained on the antigen presenting cells are recognized by T cells that provide help to B cells; and (3) the B cells are stimulated to proliferate and divide into antibody forming cells that secrete antibody against the antigen.
Most antigens only elicit antibodies with assistance from the T cells and, hence, are known as T-dependent (TD). Examples of such T-dependent antigens are tetanus and diphtheria toxoids.
Some antigens, such as polysaccharides, cannot be properly processed by antigen presenting cells and are not recognized by T cells. These antigens do not require T cell assistance to elicit antibody formation but can activate B cells directly and, hence, are known as T-independent antigens (TI). Such T-independent antigens include H. influenzae type b polyribosyl-ribitol-phosphate (PRP) and pneumococcal capsular polysaccharides.
Other differences between T-independent and T-dependent antigens are:
a) T-dependent antigens, but not T-independent antigens, can prime an immune response so that a memory response results on secondary challenge with the same antigen.
b) The affinity of the antibody for antigen increases with time after immunization with T-dependent, but not T-independent antigens.
c) T-dependent antigens stimulate an immature or neonatal immune system more effectively than T-independent antigens.
d) T-dependent antigens usually stimulate IgM, IgG1, IgG2a, and IgE antibodies, while T-independent antigens stimulate IgM, IgG1, IgG2b, and IgG3 antibodies.
T-dependent antigens can stimulate primary and secondary responses which are long-lived in both adult and in neonatal immune systems, but must frequently be administered with adjuvants. Very small proteins, such as peptides, are rarely immunogenic, even when administered with adjuvants.
T-independent antigens, such as polysaccharides, are able to stimulate immune responses in the absence of adjuvants, but cannot stimulate high level or prolonged antibody responses. They are also unable to stimulate an immature or B cell defective immune system (Mond J J., Immunological Reviews, 64:99 (1982) (Mosier D E, et al., J. Immunol., 119:1874 (1977). For T-independent antigens, it is critical to provide protective immunity against such antigens to children, especially against polysaccharides such as H. influenzae and S. pneumoniae. For T-dependent antigens, it is critical to develop vaccines based on synthetic peptides that represent the primary antigenic determinants of various pathogens.
One approach to enhance the immune response to T-independent antigens involves conjugating polysaccharides such as H. influenzae PRP (Cruse J M, Lewis R E Jr. ed., Conjugate Vaccines in Contributions to Microbiology and Immunology, Vol. 10, (1989) or oligosaccharide antigens (Anderson P W, et al., J. Immunol., 142:2464, (1989) to a single T-dependent antigen such as tetanus or diphtheria toxoid. Recruitment of T cell help in this way has been shown to provide enhanced immunity to many infants that have been immunized.
Protein-polysaccharide conjugate vaccines stimulate an anti-polysaccharide antibody response in infants who are otherwise unable to respond to the polysaccharide alone.
Conjugate vaccines are effective, but expensive to produce. See Cruse. One problem in the preparation of conjugate vaccines is removal of the unconjugated protein from the covalently linked protein. Typically, this is done by size exclusion gel filtration and requires a large and dedicated column. Houen et al. demonstrated that peptides could be coupled to protein adsorbed to solid phase aluminum adjuvants. Houen, G. et al., J. Immun. Meth., 206:125 (1997). Furthermore, they found the anti-peptide antibody response induced by these solid phase conjugates was higher than that induced by conjugates prepared in solution phase. Synthesis of these solid phase conjugates was simple because unconjugated peptide and reagents could be removed by centrifugation of the solid phase components. Importantly, since the solid phase is itself the adjuvant, there was no need for the conjugate to be removed from the solid phase scaffold on which the immunogen was synthesized. This overcomes a significant disadvantage of other solid phase synthetic approaches. But Houen et al. did not address non-peptide applications, e.g., carbohydrates.
Kossovsky discusses xe2x80x9cantigen delivery vehiclesxe2x80x9d made up of a diamond nanoparticle that is coated with cellobiose, a disaccharide, to form a xe2x80x9ccolloid surfacexe2x80x9d that would bind to a protein antigen. Kossovsky et al., Bioconj. Chem. 6(5), pp. 507-520 (1995). The antigen delivery vehicle serves to present the protein antigen to xe2x80x9cevoke a strong immunogenic responsexe2x80x9d to the protein antigen. It does not seek, and in fact it would be undesirable, to evoke any immunogenic response to the disaccharide, because cellobiose is a repeat unit of cellulose, which is a common food additive.
Thus, there remains a need in the art to simplify the preparation of solid phase dual carrier conjugate vaccines, in particular removal of free hapten, protein and polysaccharide.
Accordingly, one aspect of the present invention is to provide an improved method for preparing solid phase conjugate vaccines.
Another aspect of the invention is to provide an improved method for purifying a conjugate vaccine containing a carbohydrate.
Another aspect of the invention is to provide an improved method for preparing solid phase conjugate vaccines containing an aluminum solid phase, a protein, and a carbohydrate.
Still another aspect of the present invention is in providing an improved method for preparing a solid phase conjugate vaccines containing a hapten.
Additional aspects of the invention will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by the practice of the invention. The aspects of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
The present invention provides a novel method to prepare solid phase conjugate vaccines, wherein a protein is first adsorbed to a solid phase adjuvant and an oligosaccharide or polysaccharide is then covalently linked to the adsorbed protein.
The present invention further provides a novel method wherein a carbohydrate is first adsorbed to a solid phase adjuvant and a protein is then covalently linked to the adsorbed carbohydrate.
The present invention still further provides a method wherein the carbohydrate is activated prior to coupling to the protein.
The present invention still further provides a method to prepare solid phase conjugate vaccines, wherein a hapten is bound to a protein.
The simple, low cost process eliminates the need for chromatography and allows the rapid removal of unconjugated carbohydrate, which has been shown to inhibit the immune response.
Both the foregoing general description and the following detailed description of the invention are exemplary and explanatory only and are not restrictive of the claimed invention.